Researchers have created a transgenic mouse in which all β-actin mRNA molecules are fluorescently labeled, and used it to reveal how the transcripts are freed from storage for translation within single neurons. Their findings were published in two companion papers in the January 24 issue of Science(1,2).
Erin Schuman, managing director at the Max Planck Institute for Brain Research who was not involved with the work, called the strategy “the best possible current approach to understand the behavior of endogenous mRNAs.” Schuman co-wrote a commentary in the same issue of Science about the new studies(3).
Although β-actin is found throughout the body, it plays a particularly important role in learning and memory. In the brain, β-actin mRNA stands at the ready within neurons, waiting to change the shapes of synaptic connections in response to stimuli.
It is thought that β-actin mRNA is stored—or “masked”—inside granules stuffed with RNA and ribosomes found in the dendrites of neurons. However, using biochemical approaches that mash cells together, researchers weren’t sure how β-actin (or other masked RNA) becomes available at the right time and in the right place.
The effort to label individual mRNAs in mice was time-consuming, expensive, and risky to begin with said Robert Singer, senior author of both papers and professor and co-chair of the anatomy and structural biology department at Albert Einstein College of Medicine of Yeshiva University. It took the group six years, with one postdoc, Hye Yoon Park (now an instructor at Einstein) leading the effort. “To have one lone person working on it, and going through a lot of frustrating failures—you need a special kind of persistence to do that,” Singer added.
In the new study, the researchers created mutant mice using lentiviral transgenesis to attach large stem loops from a bacteriophage to the tail end of the β-actin gene. Those mice were crossed with another mutant mouse strain whose β-actin was tagged with bacteriophage capsid protein (which binds the stem loops) fused to green fluorescent protein. The resulting mouse mutant had about 2 million daltons worth of extra protein attached to its mRNA.
To the scientists’ surprise, the transgenic mice showed no difference in behavior or general health from wild type mice. “That’s pretty remarkable,” Singer noted. “It says something about the resilience of this biological system that it can withstand all this additional baggage on an essential molecule.”
The other paper by Singer’s group showed that in a resting state, β-actin mRNAs in neurons, unlike the same molecules in glial cells, are difficult to detect. It’s not until neurons are chemically stimulated that β-actin reveals itself—and then, only temporarily. “It’s a very elegant system that’s sort of like a flower that opens and closes very rapidly,” Singer said. “The RNA is only available for 7 or 10 minutes, and then bang, it gets sequestered again.” The results support the idea that in neurons, β-actin is normally tied up in granules found in dendritic spines, and that learning and memory-related stimuli temporarily unmask the mRNA for rapid translation precisely where the protein is needed in the cell. Ribosomes found within the granules also become available this way.
Singer’s team is working on extending their observations in vivo in mice outfitted with a cranial window. They’re also electrically stimulating acute brain slices to look more closely at how the gene is turned on.
Because β-actin is found throughout the body, the mice will be useful for studying other cell types, such as muscle and liver, and comparing how the same genes are regulated in different tissues, Singer said.
The general strategy for creating this type of mouse model could also be useful for studying other genes, although it’s “not a given that all mRNAs will tolerate the stem-loops in the 3´UTR and behave normally,” noted Schuman.
Singer’s group has, however, successfully created another mouse in which the mRNA for arc—another protein essential for memory—is labeled with a red fluorescent tag. They plan to use arc and β-actin mRNA-labeled mice to study how different mRNAs are handled by a cell or in response to particular stimuli.
1. H. Y. Park et al., “Visualization of dynamics of single endogenous mRNA labeled in live mouse,” Science, 343:422-4.
2. A. R. Buxbaum et al., “Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability,” Science, 343:419-22, 2014.
3. G. Akbalik and Schuman, E. M. “Molecular biology. mRNA, live and unmasked,” Science, 343:375-6, 2014.